SUMMARY Intervertebral disc (IVD) degeneration is a primary or secondary cause of low back pain with associated medical costs ranging from at least $20 to $100 billion annually, yet surgical treatment options do not focus on preventing degeneration or repairing discs. The high prevalence of disc degeneration in humans and rarity of disc degeneration in animal species that retain notochordal (NC) cells into maturity supports the concept that NC cells are integral to preventing degeneration and/or promoting repair. There are few studies investigating interactions between NC cells with mature IVD cells, even fewer exploring the mechanisms for these interactions, and no scientific literature on interactions between NC cells with bone mesenchymal stem cells (MSCs). The first hypothesis is that NC cells orchestrate the biology of IVDs, functioning by interacting with and influencing the functions of nucleus pulposus (NP) and annulus fibrosus (AF) cells in a dose-dependent manner that is mediated via soluble factors. The second hypothesis is that NC cells promote differentiation of MSCs towards a young NP phenotype, via the action of soluble factors. The proposed Specific Aims will test the functional relevance and mechanisms of interaction between soluble factors from NC cells with NP and AF cells (Aim 1) and with MSCs (Aim 2). The assay system will consist of 3- dimensional cell pellet cultures to maintain cell morphology, isolate soluble factor effects, and elucidate mechanisms for action. The parameters to be analyzed include cell biosynthesis and phenotypic differentiation based on IVD matrix gene expression and protein synthesis, as well as cell proliferation and apoptosis. NC, NP and AF cells are harvested from immature porcine and mature bovine IVDs as these large animal models provide a consistent source of sufficient numbers of cells required in the proposed studies. Human MSCs will be used in view of their clinical relevance. This study fills an important gap in the literature. Preliminary results are limited but compelling and demonstrate significant interactions of NC cells with mature IVD cells and evidence that NC cell soluble factors strongly increase glycosaminoglycan production in MSCs and specifically differentiate these cells towards a young NP phenotype. The exploration of a young NP phenotype along with evaluation of specific mechanisms for action in the proposed study is consistent with the exploratory/high impact objectives of the R21 mechanism. Clinical relevance involves determination of target molecules for pharmaceutical use on mature disc cells and on MSCs to prevent degenerative changes and promote cell therapy repair techniques.